PROJECT SUMMARY With the goal of devising innovative therapies to treat birth defects, diseases, and injuries that compromise the function of the temporomandibular joint (TMJ), much needs to be done to uncover mechanisms that direct the differentiation of secondary cartilage. Secondary cartilage arises after development of the primary cartilaginous skeleton and plays a critical kinetic role in the articulations and muscle attachments of the jaw. In humans, secondary cartilage that forms at the condylar and coronoid processes of the mandible is required for normal function of the TMJ. Jaw movements and associated forces are necessary to induce and maintain secondary cartilage, but how biomechanical and molecular signals become integrated to do so remains unclear. To address this question, we propose a series of experiments that leverage the distinct jaw anatomies of duck and quail embryos. Much like that found in humans, duck develop a pronounced secondary cartilage at the tendon insertion of their jaw adductor muscle on the coronoid process. An equivalent secondary cartilage is absent in quail and other species such as mice. We exploit the fact that duck form this secondary cartilage and focus on the role of neural crest mesenchyme (NCM), which produces all the cartilages and bones in the jaw skeleton. NCM also makes muscle connective tissues including ligaments and tendons. In contrast, jaw muscles are derived from mesoderm. Our published and preliminary studies show that NCM, when transplanted from quail to duck, generates quail-like pattern in the jaw skeleton and accompanying musculature, which in turn causes a loss of secondary cartilage on the coronoid process. Moreover, paralyzing muscle or blocking Transforming Growth Factor-Beta (TGF?) and Fibroblast Growth Factor (FGF) signaling also inhibits secondary chondrogenesis on the coronoid process. Thus, we hypothesize that species-specific differences in TGF? and FGF signaling, jaw architecture, and mechanical forces promote formation of secondary cartilage on the coronoid process of duck versus quail. We test our hypothesis with three Specific Aims. In Aim 1, we evaluate the extent to which TGF? and FGF signaling are NCM-mediated, and use gain- and loss-of-function approaches to determine precisely when and where these pathways induce secondary cartilage at the coronoid process. In Aim 2, we investigate the link between jaw architecture and mechanical forces at the mandibular adductor using a finite element model derived from geometric and material property studies, and tested through experimental manipulations. In Aim 3, we block mechanotransduction, modulate embryonic motility, and combine in vivo and in vitro experiments to understand how the local mechanical environment regulates molecular programs for secondary cartilage. By investigating the effects of NCM-mediated signaling, musculoskeletal anatomy, and mechanical forces on the induction of secondary cartilage, this project will offer new insights on patterning the coronoid process, which is essential to TMJ function, provide a potential means to improve tendon to bone healing, and lead to novel clinical strategies for regenerating secondary cartilage.